Toroidal mixed nanoparticle, method for preparing the same, and method for multifunctional delivery using the same

The present disclosure relates to nanotechnology, particularly to a multifunctional delivery system using a mixed nanoparticle.

Life expectancy has significantly increased. Hence, the incidence of various age-related diseases has increased and received great attentions, such as cancer, blood pressure, diabetes, hyperlipidemia, heart disease, stroke, osteoporosis and degenerative arthritis. However, some drugs show low specificity and off-target effects. For example, the use of anti-cancer drugs may be accompanied by side effects such as vomiting, nausea, fatigue and leukopenia. Therefore, the development of the drug delivery system with high specificity, good penetration, and flexibility is urgently needed.

Although many drug delivery systems have been developed in the art, there are still many problems to be solved. For example, some drug delivery systems showed low stability, insufficient elasticity (poor penetration ability). Moreover, some drug delivery systems display only little penetration effects for tumor and vessel, indicating drugs cannot reach and accumulate in tumor lesions. Furthermore, some drug delivery systems are lacking flexibility for conjugating drugs or biological agents according to actual needs. Further taking micelles as example, these systems have been used with varying degrees of success, e.g., in preclinical models, poor solubility in water miscible solvents, and relatively high critical micellar concentrations causes the micelles to fall apart rapidly when used in vivo.

Thus, there is an unmet need in the art to develop a nanoparticle for multifunctional delivery and to construct a system using the same capable of carrying drugs or biological agents, so as to solve the above problems in the field and meet the clinical needs.

Given the foregoing, the present disclosure provides a toroidal mixed nanoparticle comprising a first polymer and a second polymer interacting with the first polymer.

In at least one embodiment of the present disclosure, the toroidal mixed nanoparticle is a toroidal mixed micelle.

In at least one embodiment of the present disclosure, the toroidal mixed nanoparticle has a diameter of from about 50 nm to about 1200 nm, e.g., about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, or 1200 nm, but the present application is not limited thereto. In some embodiments, the toroidal mixed nanoparticle has a diameter of from about 50 nm to about 500 nm, 50 nm to 220 nm, or, 100 nm to 200 nm.

In at least one embodiment of the present disclosure, the toroidal mixed nanoparticle has elasticity.

In at least one embodiment of the present disclosure, the interaction between the first polymer and the second polymer is electrical property, hydrophilicity, or hydrophobicity. In some embodiments, the first polymer is an amphiphilic polymer, and the second polymer is a hydrophobic polymer, e.g., the amphiphilic polymer is d-α-tocopherol polyethylene glycol succinate, and the second polymer is a poly-γ-benzyl-1-glutamate, but the present disclosure is not limited thereto. In some embodiments, the first polymer comprises polyethylene glycol.

In at least one embodiment of the present disclosure, the toroidal mixed nanoparticle is conjugated with a drug or a bioactive agent. In some embodiments, the drug or bioactive agent is selected from the group consisting of platinum derivatives, camptothecin, doxorubicin, methotrexate, 17-(Allylamino)-17demethoxygeldanamycin (17-AAG), celecoxib, capecitabine, docetaxel, epothilone B, Erlotinib, Etoposide, GDC0941, Gefitinib, Geldanamycin, Imatinib, Intedanib, lapatinib, Neratinib, NVP-AUY922, NVP-BEZ235, Panobinostat, Pazopanib, Ruxolitinib, Saracatinib, Selumetinib, Sorafenib, Sunitinib, Tandutinib, Temsirolimus, Tipifarnib, Tivozanib, Topotecan, Tozasertib, Vandetanib, Vatalanib, Vemurafenib, Vinorelbine, Vismodegib, Vorinostat, ZSTK474, and any combination thereof, but the present disclosure is not limited thereto. In some embodiments of the present disclosure, the platinum derivatives may be dichloro(1,2-diaminocyclohexane) platinum(II) (DACHPt), but the present disclosure is not limited thereto.

The present disclosure also provides a method for preparing the toroidal mixed nanoparticle mentioned above, comprising mixing a first polymer and a second polymer to form a mixed nanoparticle, wherein the second polymer has cleavable hydrophobic groups; and removing a portion of the cleavable hydrophobic groups from the second polymer to make the second polymer charged and to form the toroidal mixed nanoparticle.

In at least one embodiment of the present disclosure, the cleavable hydrophobic groups may be benzyl group, fluorenylmethoxycarbonyl protecting group (Fmoc), tert-butoxycarbonyl protecting group (Boc), or any combination thereof, but the present disclosure is not limited thereto.

In at least one embodiment of the present disclosure, the first polymer and the second polymer are dissolved in the solution, and the mixed nanoparticle is formed via a solvent-exchange method.

In at least one embodiment of the present application, the mixed nanoparticle is reacted with acid or base to remove the portion of the cleavable hydrophobic groups from the second polymer. In some embodiments, the mixed nanoparticle is reacted with alkali, e.g., NaOH.

In at least one embodiment of the present application, the mixed nanoparticle is reacted with acid or base for about 2 hours to about 72 hours, e.g., about 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours, but the present application is not limited thereto. In some embodiments, the mixed nanoparticle is reacted with alkali for about 2 to 72 hours, 24 to 72 hours, or 24 to 36 hours.

In at least one embodiment of the present disclosure, about 10% to about 50% (e.g., about 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%), about 20% to about 50%, or about 25% to about 35% of the cleavable hydrophobic groups are removed from the second polymer, but the present application is not limited thereto.

The present disclosure also provides a method for delivering a drug or a bioactive agent to a subject in need thereof, comprising providing a pharmaceutical composition that includes the toroidal mixed nanoparticle mentioned above, an effective amount of the drug or the bioactive agent conjugated to the toroidal mixed nanoparticle, and a pharmaceutically acceptable excipient; and administering the pharmaceutical composition to the subject. In at least one embodiment of the present application, the subject suffers from cancer.

In summary, the present disclosure provides a novel and multifunctional delivery system using toroidal mixed nanoparticle. The multifunctional delivery system is easy to use and shows high stability and biosafety. With the extraordinary elasticity, the toroidal nanoparticle used in the present disclosure can penetrate the blood vessels and accumulate into tumor lesions. Multifunctional delivery system provided herein shows the flexibility for selecting the drugs or biological agents, such as anti-cancer drug, and synergistically enhances the therapeutic effects thereof.

The following embodiments are provided to illustrate the present disclosure in detail. A person having ordinary skill in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and also can implement or apply in other different embodiments. Therefore, it is possible to modify and/or alter the following embodiments for carrying out this disclosure without contravening its scope for different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein can combine with any other element or method disclosed in any embodiments of the present disclosure.

The articles “a” “an”, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The term “or” is used interchangeably with the term “and/or” unless the context clearly indicates otherwise.

As used herein, the term “about” generally referring to the numerical value meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from a given value or range. Such variations in the numerical value may occur by, e.g., the experimental error, the typical error in measuring or handling procedure for making compounds, compositions, concentrates, or formulations, the differences in the source, manufacture, or purity of starting materials or ingredients used in the present disclosure, or like considerations. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of time periods, temperatures, operating conditions, ratios of amounts, and the likes disclosed herein should be understood as modified in all instances by the term “about.”

The numeral ranges used herein are inclusive and combinable, any numeral value that falls within the numeral scope herein could be taken as a maximum or minimum value to derive the sub-ranges therefrom. For example, it should be understood that the numeral range “10-50%” comprises any sub-ranges between the minimum value of 10% to the maximum value of 50%, such as the sub-ranges from 10% to 25%, from 25% to 50%, or from 22.5% to 37.5%. In addition, a plurality of numeral values used herein can be optionally selected as maximum and minimum values to derive numerical ranges. For instance, the numerical ranges of 50 nm to 500 nm, 50 nm to 1200 nm, and 500 nm to 1200 nm can be derived from the numeral values of 50 nm, 500 nm, and 1200 nm.

As used herein, “subject” is used to mean any vertebrate including, but not limited to, humans, mammals such as deer, mule, elk, mule deer, seeking to improve a condition, disorder, or disease. However, advantageously, the subject is a mammal such as a human, or an animal mammal such as a domesticated mammal, e.g., a dog, a cat, a horse, a rat, a mouse, or the like, or a production mammal, e.g., a cow, a sheep, a pig, a deer, or the like.

The terms “comprise,” “comprising,” “include,” “including,” “have,” “having,” “contain,” “containing,” and any other variations thereof are intended used herein to cover a non-exclusive inclusion. For example, when describing an object “comprises” a limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, devices, systems, steps, or connections, etc., and should not exclude other limitations.

As used herein, “administer,” “administration,” “treatment,” “supplementation,” “injection,” or “provide” refers to a technique used to deliver a substance, i.e., stem cells into the body systemically or locally, or any combination thereof. When administering a therapeutically effective amount of the present invention parenterally or intravenously, it is generally formulated in a unit dosage form (e.g., emulsion, pills, and ointment).

The term “treating” or “treatment” refers to administration of an effective amount of a therapeutic agent to a subject in need thereof, who has the disease, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, or ameliorate the disease, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.

As used herein, the term “amphiphilic” is used herein to mean a substance containing both hydrophilic or polar (water-soluble) and hydrophobic (water-insoluble) groups.

The term “hydrophilic” refers to the tendency of a material to disperse freely in aqueous media. A material is considered hydrophilic if it prefers interacting with other hydrophilic material and avoids interacting with hydrophobic material. “Hydrophilicity” used herein may be a relative term, i.e., the same molecule could be described as hydrophilic or not depending on what it is being compared to. In at least one embedment of the present application, the polymer has cleavable hydrophilic groups that can be removed via a chemical reaction such as acid or base treatment, and the hydrophilicity of the polymer is reduced when part of the hydrophilic groups is removed therefrom; however, the polymer still belongs to the hydrophilic compound as defined herein. In some embodiments, hydrophilic molecules are polar and/or charged and have good water solubility, e.g., are soluble up to 0.1 mg/mL or more, but the present disclosure is not limited thereto.

As used herein, the term “hydrophobic” refers to the tendency of a material to avoid contact with water. A material is considered hydrophobic if it prefers interacting with other hydrophobic material and avoids interacting with hydrophilic material. “Hydrophobicity” used herein may be a relative term, i.e., the same molecule could be described as hydrophobic or not depending on what it is being compared to. In at least one embedment of the present application, the polymer has cleavable hydrophobic groups that can be removed via a chemical reaction such as acid or base treatment, and the hydrophobicity of the polymer is reduced when part of the hydrophobic groups is removed therefrom; however, the polymer still belongs to the hydrophobic compound as defined herein. In some embodiments, hydrophobic molecules are nonpolar and/or uncharged and have poor water solubility, e.g., are insoluble down to 0.1 mg/mL or less, but the present disclosure is not limited thereto.

The term “delivery system” refers to a method or process of administering a pharmaceutical compound or bioactive agent to achieve a therapeutic effect in subject in need thereof.

The term “particle” refers to a nano- or micro-sized supramolecular structure comprised of an assembly of molecules. For example, in some embodiments, the amphiphilic block polymer forms a particle in aqueous solution. In some embodiments, particle formation by the amphiphilic block polymer is dependent on pH or temperature.

Materials and Animals

Polymers, including d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and poly(γ-benzyl-1-glutamate) (PBLG), and the anticancer reagent, dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt), were all purchased from Sigma-Aldrich (St. Louis, M.O., U.S.A.). The organic solvents, N,N-dimethylacetamide (DMAc) and dimethylformamide (DMF), were both obtained from Duksan Pure Chemical Co., LTD. (Gyeonggido, South Korea), and the o-phenylenediamine (OPDA) reagent used to quantify the platinum reagent was purchased form Alfa Aesar (Ward Hill, M.A., U.S.A.). The dialysis bags were acquired from Rainbow Biotechnology Co., LTD. (Taipei City, Taiwan). The staining reagent for preparing the transmission electron microscopic samples, sodium phosphotungstate (PTA), and the analytical reagents, including dimethyl sulfoxide (DMSO-d) for hydrogen nuclear magnetic resonance (H-NMR) measurements and the potassium bromide (KBr) for Fourier-transform infrared spectroscopic (FT-IR) analysis, were also purchased from Sigma-Aldrich. Sodium hydroxide (NaOH) for nanoparticle preparation and the sodium chloride (NaCl) for stability testing and drug releasing profiles were, respectively, acquired from Uniregion BioTech Inc. and Vetec of Sigma-Aldrich. The dialysis bags and PD-10 desalting columns for purification were, respectively, purchased from Merck Millipore (Burlington, M.A., U.S.A.) and GE Healthcare Life Science (Uppsala, Sweden). The fluorescent dyes, including 5/6-carboxyfluorescein succinimidyl ester (FITC-NHS ester), Cellmask Orang and CellTracker™ Red CMTPX dye were obtained from Thermo Fisher Scientific Inc. (Waltham, M.A., U.S.A.). The cell media, including Dulbecco's modified Eagle's medium (DMEM), McCoy's 5a medium, fetal bovine serum (FBS), and penicillin-streptomycin solution, for the cell culture were acquired from Cytiva (Marlborough, M.A., U.S.A.). The edocytosis inhibitors including amiloride and methyl-β-cyclodextrin (methyl-β-CD) were purchased from Merck and Co. (Rahway, N.J., U.S.A.) and sucrose were obtained from J.T. Baker (Radnor, P.A., U.S.A.). The p-Slide I Luer flow channel chips, polymer coverslip bottom cell-cultured dishes and bioinert cell-cultured dishes were all purchased from ibidi GmbH (Gewerbehof, Grafelfing, Germany). The 96-well microplate for culturing tumor spheroids were purchased from Corning Inc. (Corning, N.Y, U.S.A.). The reagents for bio-TEM observations, including 2.5% glutaraldehyde, 1% osmium tetroxide, uranyl acetate, and lead citrate, were kindly provided by Nautiagene (Taipei City, Taiwan, R.O.C.). Besides, the reagents for H & E staining and for paraffin embedment were kindly provided from Professor Jiunn-Wang Liao.

The animal tests were approved by Institutional Animal Care and Use Committee (IACUC) in China Medical University (IACUC approval number: CMUIACUC-2020-058) and the BALB/c nude mice for our animal tests were provided by National Laboratory Animal Center (Taipei City, Taiwan, R.O.C.). The materials in our animal tests, including Matrigel and isoflurane were respectively obtained from Merck and Panion & BF Biotech. Inc. (Taiwan). The fluorescence dye, cyanine 5.5 NHS ester was acquired from Lumiprobe (Wan Chai, Hong Kong).

The formalin for tissue fixation was purchased from Sigma-Aldrich. The 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium, the antibodies for frozen tissue staining, including anti-CD34 and anti-45 primary antibodies and the fluorescent secondary antibodies, including Alexa Fluor®488 and 555-labeled anti-rabbit IgG secondary antibodies were obtained from Abcam PLC (Cambridge, U.K.).

Preparation and Characterization of the DACHPt-Loaded Toroidal Mixed Micelles

The TPGS (2 mg) and PBLG (6 mg) polymers were dissolved in DMAc (16 mL) and assembled into polymeric mixed micelles using the solvent exchange method. The fabricated mixed micelles were concentrated using 3000 RPM. ultrafiltration (M.W.C.O. 10K) for 10 min. The concentrated mixed micellar solution was placed into a sample vial, and 0.337 mL of an NaOH aqueous solution (0.5 N) was dropped into the sample vial. The sample vial was then incubated at 25° C. under stirring for an appropriate reaction period. Afterwards, the solution was placed into dialysis bags (M.W.C.O. 6-8 k) and dialyzed against deionized water overnight. The solution was taken from the dialysis bags, and 0.272 mL of DACHPt aqueous complexes was added. The solution was further reacted for a period at 25° C. under stirring. Once the reaction was terminated, excess DACHPt aqueous complexes were removed with 3000 RPM ultrafiltration (M.W.C.O. 30k) for 10 min. Particle sizes and zeta potentials were measured with dynamic light scattering (DLS) (ZS 90, Malvern, U.K.).

The morphologies of the DACHPt-loaded toroidal mixed micelles were observed with a transmission electron microscope (TEM) (JEM-2100F, JEOL Ltd., Japan) and an atomic force microscope (AFM) (Dimension Icon, Bruker, M.A., U.S.A.). For TEM observations, 10 μL of the sample solutions were dropped onto carbon-coated copper grids and a few minutes later, the solutions were removed from the grids. Afterwards, a staining dye containing 1% sodium phosphotungstate (10 μL) was dropped onto the grids for 1 min. After removal of the excess staining dye, the grids were dried and stored at 25° C. for observations. The observations were conducted with a field emission transmission electron microscope (FE-TEM) under an accelerated voltage of 200 kV. Simultaneously, the element distribution of the DACHPt-loaded toroidal mixed micelles was analyzed via energy-dispersive X-ray spectroscopy (EDS) and the INCA software.

The nanostructures were also characterized with an AFM and analyzed with Nanoscope Analysis software. The sample solutions were dropped onto a silicon wafer that was previously air washed and superficially treated with plasma for 1 min. After drying in a vacuum oven, the silicon wafer was moved to AFM for topological observations and quantitative nanomechanical (QNM) measurements. Monocrystal silicon tips (Brucker, Bruker, M.A., U.S.A.) with a nominal spring constant (kN) of 0.7 N/m were selected as the cantilevers. The Young's modulus was analyzed every 10 points in the particles and calculated with the Hertzian contact model.

Drug-Loading Contents, Efficiency, and Stability, Releasing Profiles

To assess the drug contents and loading efficiency of the DACHPt-loaded toroidal mixed micelles, the prepared DACHPt-loaded toroidal mixed micelles were freeze-dried. They were then weighed and redispensed in 1 mL of 20% NaCl solution. After a 24 h reaction, released platinum was detected and quantified via OPDA methods (Zhang, Weiqi, and Ching-Hsuan Tung. “Redox-responsive cisplatin nanogels for anticancer drug delivery.”54.60 (2018): 8367-8370). Briefly, equal volumes of the DACHPt-loaded TM and OPDA solutions (1.2 mg/mL in DMF) were homogeneously blended, and the mixture was bathed at 80° C. for 10 min. As the mixed solution cooled down to 25° C., the platinum concentration was detected at 703 nm with a UV-vis spectrophotometer (Lambda 265, PerkinElmer, M.A., U.S.A.).

The stability tests and drug releasing profiles were conducted for the comparison of DACHPt-loaded polymeric spherical and toroidal mixed micelles. In advance, the DACHPt-loaded spherical polymeric mixed micelles were prepared as following: the polymers, including TPGS (2 mg) and PBLG (6 mg) were dissolved into DMAc (16 mL) along with 2 mg of DACHPt regents. After dialysis against deionized water, the DACHPt-loaded spherical polymeric mixed micelles spherical polymeric mixed micelles were filtered to remove the excess DACHPt and stored at 4° C. until uses. Their morphology was observed using a transmission electron microscope and physical properties including particle sizes, distributions and their mechanical properties (Young's modulus) were measured following the methods of DACHPt-loaded toroidal mixed micelles. A stability test was then conducted as follows: DACHPt-loaded spherical polymeric mixed micelles (0.5 mL) and DACHPt-loaded toroidal mixed micelles (0.5 mL) were mixed with an equal volume of deionized water. DACHPt-loaded toroidal mixed micelles (0.5 mL) were then additionally blended with equal volume of phosphate buffering saline (PBS) (0.5 mL). At a predetermined time, the DACHPt-loaded spherical polymeric mixed micelle and toroidal mixed micelle sizes were analyzed using DLS to study the stability. The releasing profiles of the DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were also determined upon incubation at a mimetic physiological environment. DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were first blended with an equal volume of deionized water or PBS, and the solutions were placed into dialysis bags (MWCO 6-8 k). The dialysis bags were then placed in deionized water or PBS at 37° C. and shaken. At a predetermined time, the released DACHPt solution was collected and quantified using OPDA methods, as mentioned above.

Internalization, Tumor Penetration Assessment in Static State and Cytotoxicity

To assess and compare the internalization of the DACHPt-loaded spherical polymeric mixed micelles and the DACHPt-loaded toroidal mixed micelles, the fluorescent dye (FITC) was conjugated onto these two nanoparticles, respectively. In brief, the amino capping TPGS was first modified by reacting with cysteine via an ester linkage. The modified TPGS-NH(2 mg), PBLG (6 mg), and anticancer potent reagent DACHPt (2 mg) were then weighed and dissolved in DMAc (16 mL). After dialysis against deionized water, the DACHPt-loaded polymeric mixed micelles were filtered to remove the excess DACHPt. Afterwards, the fluorescent dye, FITC-NHS ester, was dissolved in DMSO (1 mg/mL), and the solution was blended with DACHPt-loaded spherical polymeric mixed micelles at 25° C. Twenty-four hours later, the solution was passed through a PD-10 desalting column to eliminate the excess fluorescent dye, forming FITC-labeled DACHPt-loaded spherical polymeric mixed micelles. The FITC-labeled DACHPt-loaded toroidal mixed micelles were also prepared following the same procedure. Modified TPGS-NHwas involved in the toroidal mixed micelle preparation described above, and the amino groups of TPGS-NHon the toroidal mixed micelles further reacted with the fluorescent dye, FITC. After passing through the PD-10 desalting column, the FITC-labeled DACHPt-loaded toroidal mixed micelles were complete.

The internalization of FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles was evaluated with the murine macrophage cell line RAW 264.6 and HCT116 human colon cancer cells. The RAW 264.7 and HCT116 cells (1×10cells per well) were seeded on a 6-well plate and incubated, respectively, with DMEM and McCoy's 5a cell culturing medium at 37° C. with a 5% COsupply. As the cells were attached, FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were independently treated with cells. At 1, 3 and 6 h post-incubation, the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were removed, and the cells were twice washed with PBS. The interior fluorescence of these cells was analyzed via flow cytometry (BD FACSCanto, Becton, Dickinson and Company, East Rutherford, N.J., U.S.A.). The internalization was also observed in real-time by a high speed confocal system (Andor Dragonfly, Oxford Instrument plc, Oxfordshire, U.K.). Murine macrophage RAW 264.7 cells and HCT116 human colon cancer cells (1×10cells) were seeded on a coverslip-bottom dish (Ibidi GmbH, Grafelfing, Germany). After the cells were attached onto the dish, they were incubated with CellMask Orange (1 μM) for 10 min to stain the cell membranes. Afterwards, FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were independently treated with RAW 264.7 and HCT116 cells, and the cells were simultaneously observed with the confocal system for 2 min. The fluorescence of the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles or toroidal mixed micelles and the cell membrane were, respectively, detected at excitation wavelengths of 488 and 554 nm and emission wavelengths of 520 and 567 nm. To confirm the morphology of DACHPt-loaded toroidal mixed micelles on the surface of the HCT116 cells, the cells after 1 h incubation with DACHPt-loaded toroidal mixed micelles were fixation by a series of dehydration. The cells and DACHPt-loaded toroidal mixed micelles were observed using SEM.

In addition, the endocytosis pathway of DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles into human colon cancer cells HCT116 was also determine using flow cytometry. The HCT116 cells (1×10cells per well) were seeded onto each well on a 6-well plate. Various endocytic inhibitors, including amiloride hydrochloride (1 mM), methyl-β-cyclodextrin (20 mM) and hypertonic sucrose (0.25 M) were treated with HCT116 cells for 30 min; afterwards, the cells were washed thrice with PBS and co-cultured with the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles. Two hours later, the cells were washed with PBS, collected and analyzed with flow cytometry. The fluorescence of the cells was compared to that of the cells only treated with FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles for 2 h.

The cell adhesion was also evaluated. The human colon cancer cells HCT116 (1×10cells/mL) were seeded on each well in a 6-well plate. When the cells were attached, the cells were incubated at 4° C. for 30 min. After pre-cooling the cells, the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles and toroidal mixed micelles were treated with the cells for 0.5 h and afterwards, the cells were washed with PBS twice and cultured at 37° C. Two hours later, the cells were washed with PBS twice and collected. The cellular fluorescence was determined by a flow cytometry.

The penetration into cancer cell spheroid in static state was also under a short-termed observation. Human colon-cancer cells HCT116 (1×10cells/mL) were seeded on a bioinert cell dish and incubated with McCoy's 5a medium at 37° C. with a 5% COsupply. Three days later, as the cells clustered together, the cells were further incubated with FITC-labeled DACHPt-loaded spherical polymeric mixed micelles or toroidal mixed micelles. The cells as well as the fluorescence was observed using the real-time by a high speed confocal system. The fluorescence of the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles or toroidal mixed micelles were detected at excitation wavelengths of 488 and emission wavelengths of 520 nm. In addition, the tumor penetration was also tracked for 6 h. Firstly, the human colon-cancer cells HCT116 (5×10cells) were stained with CellTracker™ Red CMTPX Dye and seeded on a 96-well ultra-low attachment plates (Corning Inc., Corning, NY, USA). Three days later, as the cell spheroids formed, the FITC-labeled DACHPt-loaded spherical polymeric mixed micelles or toroidal mixed micelles were treated. The fluorescence of the cell tracker and FITC was detected using IncuCyte S3 cell tracking system (Essen BioScience Inc., Ann Arbor, MI, USA).

The cytotoxicity of the DACHPt and DACHPt-loaded toroidal mixed micelles toward HCT116 human colon cancer cells was evaluated with an MTT assay. Various concentrations (0.16-20 mg/mL) of the DACHPt aqueous complexes and DACHPt-loaded toroidal mixed micelles that were previously adjusted based on the DACHPt concentration were treated with HCT116 human colon cancer cells for 24 h. Cell viability was then determined using an MTT assay.

In Vitro Behaviors Under Flow Conditions